Tang Jie, Li Ran, Jiang Tingyu, Lv Jiachen, Jiang Yuwei, Zhou Xingjian, Chen Hong, Li Meiliang, Wu Aimin, Yu Bing, Takala Timo M, Saris Per E J, Li Shuhong, Fang Zhengfeng
Key Laboratory for Animal Disease-Resistance Nutrition of China Ministry of Education, Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China.
Key Laboratory of Agricultural Product Processing and Nutrition Health (Co-Construction by Ministry of Agriculture and Rural Affairs of China and Sichuan Province), College of Food Science, Sichuan Agricultural University, Ya'an 625014, China.
Microorganisms. 2024 Nov 25;12(12):2414. doi: 10.3390/microorganisms12122414.
In this study, the probiotic yeast was engineered to secrete the antiviral lectin griffithsin. Twelve genetic tools with the griffithsin gene were cloned into the vector pSF-TEF1-URA3 and introduced into . In the recombinant strains, a 16.9 kDa band was detected using SDS-PAGE and further recognized by griffithsin antibody with Western blotting. strains FM, FT, HC, and HE with a high yield of griffithsin were acquired for property characterization in vitro. The four recombinant strains displayed a similar growth pattern to that of the control strains, while their morphological characteristics had changed according to scanning electron microscopy. In simulated gastrointestinal digestive fluids, the survival rates of FM, FT, and HC were significantly decreased (86.32 ± 1.49% to 95.36 ± 1.94%) compared with those of the control strains, with survival rates between 95.88 ± 0.00% and 98.74 ± 1.97%. The hydrophobicity of FM, the strain with the highest griffithsin production, was significantly increased to 21.89 ± 1.07%, and it exhibited a reduced auto-aggregation rate (57.64 ± 2.61%). Finally, Vero cells infected with porcine epidemic diarrhea virus (PEDV) were used to evaluate the strains' antiviral activity, and the rate at which FM inhibited PEDV reached 131.36 ± 1.06%, which was significantly higher than that of the control group.
在本研究中，对益生菌酵母进行基因工程改造，使其分泌抗病毒凝集素格里菲斯素。将12种携带格里菲斯素基因的遗传工具克隆到载体pSF-TEF1-URA3中，并导入[具体受体]。在重组菌株中，通过SDS-PAGE检测到一条16.9 kDa的条带，并用格里菲斯素抗体进行蛋白质印迹进一步确认。获得了高产格里菲斯素的FM、FT、HC和HE菌株用于体外特性表征。这四种重组菌株的生长模式与对照菌株相似，但其形态特征根据扫描电子显微镜观察有所变化。在模拟胃肠道消化液中，与对照菌株相比，FM、FT和HC的存活率显著降低（从86.32±1.49%降至95.36±1.94%），对照菌株的存活率在95.88±0.00%至98.74±1.97%之间。产格里菲斯素量最高的FM菌株的疏水性显著增加至21.89±1.07%，且其自聚集率降低（57.64±2.61%）。最后，用感染猪流行性腹泻病毒（PEDV）的Vero细胞评估这些菌株的抗病毒活性，FM菌株对PEDV的抑制率达到131.36±1.06%，显著高于对照组。
